We want to understand the mechanisms that underlie the maturation of the antibody response to antigen. Central to such maturation are somatic hypermutation (SHM) and class switch DNA recombination (CSR). In SHM, point-mutations accumulate in immunoglobulin (Ig) heavy (H) and light chain V(D)J regions, thereby providing the substrate for selection of higher affinity mutants by antigen. In CSR, the constant (C)[unreadable]chain gene is replaced by C?, Ca or Ce, resulting in new biological effector functions, such as extravascular clearance of pathogens (IgG) or mucosal protection (IgA, IgE). In this proposal, we will address the mechanisms of CSR. We argue that the conserved S region AGCT motif recruits specific trans-factors, particularly 14-3-3 adaptor proteins, to mediate CSR, which proceeds through generation of activation-induced cytidine deaminase (AID)- processed double-strand DNA break (DSBs). We also argue that such DSBs are resolved through a process entailing intervention of DNA mismatch repair (MMR) proteins, including Mlh3, the last complement of the mammalian set of MMR proteins, and error-prone translesion synthesis (TLS) DNA polymerases, such as polymerase (pol) ? and the recently characterized pol ?, eventually leading to S-S DNA junctions involving nucleotide "microhomologies" or "insertions". Finally, we hypothesize that germline IH-CH transcription and CSR are upregulated by the IgH 3' enhancer (3'Ea) elements as induced by HoxC4, a highly conserved homeodomain protein that, as we showed, binds to and induces the human 3'Ea hs1,2 enhancer. To test our hypotheses, we propose to: (i) address the role of S region AGCT motifs in recruiting specific trans- factors, namely 14-3-3 adaptors, and outline the role of these proteins in CSR; (ii) define the role of the MMR proteins Mlhs, Pmsi and other MutL homologs, and TLS DNA pol ? and pol ?, (Revs catalytic subunit) in CSR; and (iii) address the role of the phylogenetically conserved HoxC4 homeodomain protein in the regulation of CSR through induction of the IgH 3'Ea enhancers and, possibly, modulation of AID expression using our newly derived hoxC4-/- mice. These experiments will be performed using novel mice and reagents, including 14-3-3?-/-, mlh3-/-, prns1-/-, pol ? -/-, conditional rev3?/?, hypomorphic rev3hypo/-, and hoxC4-/- knockout mice, and our human monoclonal B cell lines that undergo CSR at a high rate. Relevance to public health: By unveiling mechanisms underlying CSR, these studies will help understand how antibodies to bacteria and viruses or pathogenic autoantibodies, such as those occurring in patients with autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis and type I diabetes, are generated by the human body, eventually leading to the development of better vaccines and therapeutics. [unreadable] [unreadable] [unreadable]